dntp  (TaKaRa)

 
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    dNTP
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    Catalog Number:
    639125
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    Structured Review

    TaKaRa dntp

    https://www.bioz.com/result/dntp/product/TaKaRa
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dntp - by Bioz Stars, 2019-12
    75/100 stars

    Related Products / Commonly Used Together

    mgcl2
    kapa taq polymerase
    kapa taq buffer

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    Related Articles

    Clone Assay:

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. After purification of the digested promoter candidates (E.Z.N.A.™ Cycle Pure Kit 200, Omega Bio-tek, Inc., USA) and the vector enzyme digestion products, the ligation was performed using T4 ligase (Thermo Fisher Scientific, USA) according to the instructions of the manufacturer and transformed into E . coli DH5α.

    Amplification:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Multiple PCR reactions were prepared to allow amplification of the total harvested gDNA from a 1000x cell coverage for each sample. .. For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The fragment of Cx32 promoter −651 to +84 (transcription start site as +1, total 735 base pairs) was PCR amplified from human genomic DNA by the following primers with restriction enzyme BglII and NheI sites added (underlined respectively): Forward, 5′-GAAGATCTAATGGTTAGCCTTTGCTTTC-3′ and reverse, 5′-CTAGCTAGCTATGGTCTCATGTCTGTGCAGG-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc., Otsu Japan), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min.

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The complete coding sequence of GATA-3 was PCR amplified from human cDNA by using the following primers with additional NheI and XhoI digestion sites added (underlined respectively): Forward, 5′-CGCTAGCGATGGAGGTGACGGCGGA-3′ and reverse, 5′-GCCTCGAGCTAACCCATGGCGGTGAC-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc.), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min.

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl. .. The reverse transcripts obtained were used as PCR templates.

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: Paragraph title: DNA extraction and polymerase chain reaction (PCR) amplification ... The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: cDNAs encoding the complete open reading frame (ORF) of zebrafish mek1/2 and erk1 were obtained by polymerase chain reaction (PCR) amplification using gene-specific primers according to the NCBI’s zebrafish EST database. .. PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The four intron SNPs (rs3087404[A/G], rs2029167 [A/G], rs2029166 [C/T] and rs7296239 [C/T]) of SMUG1was detected by Modified polymerase chain reaction-mismatch amplification (MA-PCR) (As described in detail previously ). .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: The six intronic SNPs of the DUT gene were detected by a modified polymerase chain -mismatch amplification (MA-PCR) reaction as described previously . .. In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: The six intronic SNPs of the DUT gene were detected by a modified polymerase chain -mismatch amplification (MA-PCR) reaction as described previously . .. In brief, the PCR was performed in a 30 µL r eaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). .. The PCR protocol was 5 min at 94 °C for initial denaturing, followed by 34 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min.

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: To simplify construction and utilize a strategy that did not rely on restriction enzymes, we used prolonged-overlap-PCR to replace the bgaB gene of pDL with gfpmut1 [ ]. .. Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. 2000 bp upstream of the start codon of the target protein were amplified as the promoter candidates.

    Article Title: Identification of a pathogenic mutation in a Chinese pedigree with polycystic kidney disease
    Article Snippet: In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total. .. In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total.

    Mass Spectrometry:

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). .. After that, 40 μL of each PCR product from the pooled DNA was bi-directionally sequenced using the ABI3730XL (Applied Biosystems, Foster City, CA, USA), and the sequences were aligned to the bovine reference sequences (UMD3.1) using BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) to identify potential SNPs.

    Synthesized:

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: The H19 -specific primers used for the reaction were synthesized by 216 bp fragments of imprinted gene H19 which contained 18 CpG loci (genbank accession no. AFl 25183; nucleotides 7881-809): H19 forward, 5′-TGGGTATTTTTGGAGGTTTTTTT-3′, and reverse, 5′-ATAAATATCCTATTCCCAAATAA-3′ (Beijing Liuhe Huada Genetic Science and Technology Co., Ltd., Beijing, China). .. The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling
    Article Snippet: Total RNA was extracted using Trizol reagent (Invitrogen Corporation, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized using the Goscript reverse transcription system (Promega Corporation, WI, USA). .. RT-PCR was performed in 0.2-mL tubes containing 10× Taq buffer, dNTP, Taq DNA polymerase (Takara, Japan), and 25 pmole of TPH1 primer (forward 5′-ATGATTGAAGACAATAAGGAG-3′ reverse 5′-AAGTTTTTGAGATACTCTCTG-3′) or GAPDH primer (forward 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse 5′-CAAAGTTGTCATGGATGACC-3′).

    Article Title: Enterovirus genotypes causing hand foot and mouth disease in Shanghai, China: a molecular epidemiological analysis
    Article Snippet: The RNA was dissolved in 20 μL DEPC (Diethypyrocarbonate) water. cDNA was synthesized using 4 μL of extracted RNA, 100 μmol of random primers, and 2.5 U of reverse transcriptase (PrimeScript TM RT kit, Takara, Dalian, China). .. The first PCR step was performed in a reaction volume of 25 μL, including 2 μL cDNA, 0.5 μmol each of outer primers (EV1F: 5′-CGGCCCCTGAATGCGGC-3′, EV1R: 5′-CACCGGATGGCCAATCCA-3′), 50 μmol dNTP, and 0.75U of ExTaq DNA polymerase (Takara, Dalian, China).

    Cytometry:

    Article Title: Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells
    Article Snippet: U251 cells were divided and treated according to the protocol described in the study using flow cytometer assay. .. The qRT-PCR was performed in a 25 μL total volume comprising 2 μL of 10× PCR buffer, 2 μL of Takara dNTP (2.5 mM), 1 μL of 5′ primer (20 pmol/μL), 1 μL of 3′ primer (20 pmol/μL), 0.5 μL of Takara TaqE (5 U/μL), 2.5 μL of MgCl2 (25 mM), 11 μL of DEPC-treated water, and 5 μL of cDNA on the PTC-100 (MJ Research, Inc., Watertown, MA, USA).

    Quantitative RT-PCR:

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Paragraph title: RT-qPCR verification ... Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: MiR-494 acts as a tumor promoter by targeting CASP2 in non-small cell lung cancer
    Article Snippet: Paragraph title: RNA extraction and quantitative RT-PCR ... Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl.

    Article Title: Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells
    Article Snippet: Reverse transcription was performed with a 20 μL total volume comprising 2 μL of extracted RNA, 1 μL of Takara Oligo dTl8 , 2 μL of Takara dNTP (10 mM), 0.5 μL of Takara RNasin, 1 μL of Takara AMV, 4 μL of 5× AMV Buffer, and 9.5 μL of DEPC-treated water. .. The qRT-PCR was performed in a 25 μL total volume comprising 2 μL of 10× PCR buffer, 2 μL of Takara dNTP (2.5 mM), 1 μL of 5′ primer (20 pmol/μL), 1 μL of 3′ primer (20 pmol/μL), 0.5 μL of Takara TaqE (5 U/μL), 2.5 μL of MgCl2 (25 mM), 11 μL of DEPC-treated water, and 5 μL of cDNA on the PTC-100 (MJ Research, Inc., Watertown, MA, USA). .. The sequences of the specific primers used in the research were as follows: caspase-3 – forward: 5′-AAACAGTATGCCGACAAG-3′ and reverse: 5′-GAGGGAAATACAGTACCAAA-3′; caspase-8 – forward: 5′-AAAGGGAACTTCAGACACC-3′ and reverse: 5′-CAGCAGGCTCTTGTTGAT-3′; caspase-9 – forward: 5′-CGAACTAACAGGCAAGCA-3′ and reverse: 5′-TTCACCTCCACCATGAAAT-3′; β-actin – forward: 5′-CGTCTGGACCTGGCTGGCCGGGACC-3′ and reverse: 5′-CATGAAGCATTTGCGGTGGACGATG-3′.

    SYBR Green Assay:

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following RNA extraction, the expression levels of the miRNA let-7f-5p, miR-10a-5p, miR-125a-5p, miR-144-3p, miR-195-5p and miR-203 in each sample was determined using RT-qPCR verification with stem-loop primers, using SYBR Green dye and U6 as the internal reference. .. Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling
    Article Snippet: Quantitative mRNA analysis was performed using a QuantiTect SYBR Green PCR kit (Qiagen, CA, USA). .. RT-PCR was performed in 0.2-mL tubes containing 10× Taq buffer, dNTP, Taq DNA polymerase (Takara, Japan), and 25 pmole of TPH1 primer (forward 5′-ATGATTGAAGACAATAAGGAG-3′ reverse 5′-AAGTTTTTGAGATACTCTCTG-3′) or GAPDH primer (forward 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse 5′-CAAAGTTGTCATGGATGACC-3′).

    Incubation:

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: For reverse transcription, the mixture was prepared as follows: All primers (Guangzhou RiboBio Co., Ltd., Guangzhou, China) were added to 24 µl RNase free water, mixed, incubated for 10 min at 70°C and subsequently kept in an ice bath for 2 min. Primer sequences were as follows: Forward, 5′-CTTGTCCTTCATTCCACCGCA-3′ and reverse, 5′-TGCCGCCTGAACTTCACTCC-3′. .. Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Next, poly-C tailing was initiated by mixing 28 μl of end-repaired DNA, 1 μl of 10× EX buffer (Takara, supplied with RR006A) and 1 μl of 1 mM dCTP (NEB, N0446S) and then denaturing the DNA. .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer. .. The anchor primer was designed to have nine consecutive Gs plus an H (which represents the nucleotides A, T or C, but not G) at the 3′-end to promote proper annealing at the beginning of the poly-C tail (as illustrated in Figure ).

    Article Title: Rapid and Specific Detection of All Known Nipah virus Strains’ Sequences With Reverse Transcription-Loop-Mediated Isothermal Amplification
    Article Snippet: The RT-LAMP reaction was performed in a 25.0 μl volume, containing 0.5 mM betaine (5 mM stock, Sigma-Aldrich Inc., St Louis, MO, United States), 1.4 mM each dNTP (10.0 mM stock, TaKaRa Bio Inc., Clontech Laboratories, Inc., Dalian, China), 2.5 μl of 10× ThermoPol Reaction Buffer [(1 × ThermoPol Reaction Buffer contains 20.0 mM Tris–HCl, pH 8.8; 10.0 mM KCl; 2.0 mM MgSO4 ; 10.0 mM (NH4 )2 SO4 ; 0.1% Triton X-100) New England Biolabs Inc.], 6.0 mM MgSO4 (New England Biolabs Inc.), 32 pM each of forward inner primer (FIP) and backward inner primer (BIP), 15 pM each of forward loop (LF) and backward loop (LB) primers (or replaced with an equal volume of nuclease-free water), 4 pM each of forward outer (F3) and backward outer (B3) primers, 8.0 U of Bst DNA polymerase large fragment (New England Biolabs Inc.), 7.5 U of WarmStart RTx reverse transcriptase (New England Biolabs Inc.; only added to the reaction system when the template was RNA), and 2.0 μl of pseudovirus RNA (≥50 pg/μl). .. The RNA from the cell extract transfected with pHAGE-CMV-MCS-IZsGreen vector and nuclease-free water were used as the negative controls, and the plasmids containing the three N genes were used as the positive controls (mixed without 7.5 U of WarmStart RTx reverse transcriptase).

    Expressing:

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: Paragraph title: GATA-3 expression vector ... The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc.), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min.

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following RNA extraction, the expression levels of the miRNA let-7f-5p, miR-10a-5p, miR-125a-5p, miR-144-3p, miR-195-5p and miR-203 in each sample was determined using RT-qPCR verification with stem-loop primers, using SYBR Green dye and U6 as the internal reference. .. Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: MiR-494 acts as a tumor promoter by targeting CASP2 in non-small cell lung cancer
    Article Snippet: Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl. .. Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl.

    Modification:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech).

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: Bisulfite modification of the DNA was conducted using an EpiTect Bisulfite kit (cat. no. 59104; Qiagen China Co., Ltd., Shanghai, China) in accordance with the manufacturer's protocol, and the DNA samples were then preserved at −20°C. .. The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The four intron SNPs (rs3087404[A/G], rs2029167 [A/G], rs2029166 [C/T] and rs7296239 [C/T]) of SMUG1was detected by Modified polymerase chain reaction-mismatch amplification (MA-PCR) (As described in detail previously ). .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: The six intronic SNPs of the DUT gene were detected by a modified polymerase chain -mismatch amplification (MA-PCR) reaction as described previously . .. In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: The six intronic SNPs of the DUT gene were detected by a modified polymerase chain -mismatch amplification (MA-PCR) reaction as described previously . .. In brief, the PCR was performed in a 30 µL r eaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Transformation Assay:

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. The enzyme digestion sites in the vector 5’ and 3’ ends were BamHI and KpnI (BamHI/EcoRI, EcoRI/KpnI).

    Countercurrent Chromatography:

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: cDNAs encoding the complete open reading frame (ORF) of zebrafish mek1/2 and erk1 were obtained by polymerase chain reaction (PCR) amplification using gene-specific primers according to the NCBI’s zebrafish EST database. .. PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Flow Cytometry:

    Article Title: Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells
    Article Snippet: U251 cells were divided and treated according to the protocol described in the study using flow cytometer assay. .. The qRT-PCR was performed in a 25 μL total volume comprising 2 μL of 10× PCR buffer, 2 μL of Takara dNTP (2.5 mM), 1 μL of 5′ primer (20 pmol/μL), 1 μL of 3′ primer (20 pmol/μL), 0.5 μL of Takara TaqE (5 U/μL), 2.5 μL of MgCl2 (25 mM), 11 μL of DEPC-treated water, and 5 μL of cDNA on the PTC-100 (MJ Research, Inc., Watertown, MA, USA).

    Ligation:

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. The enzyme digestion sites in the vector 5’ and 3’ ends were BamHI and KpnI (BamHI/EcoRI, EcoRI/KpnI).

    Cell Culture:

    Article Title: MiR-494 acts as a tumor promoter by targeting CASP2 in non-small cell lung cancer
    Article Snippet: We used Trizol (Invitrogen, USA) regent to isolate total RNA from cultured cells according to the manufacturer’s protocol; 2 µg of total RNA were reverse transcribed with random primer. .. Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl.

    Polymerase Chain Reaction:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Paragraph title: PCR recovery of sgRNA sequences from gDNA ... For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech).

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: All first round PCRs were pooled and a fraction was used as template for the second round PCR. .. The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 55C – 10 sec, 72C – 20 sec), 1x (68C – 2 min).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The fragment of Cx32 promoter −651 to +84 (transcription start site as +1, total 735 base pairs) was PCR amplified from human genomic DNA by the following primers with restriction enzyme BglII and NheI sites added (underlined respectively): Forward, 5′-GAAGATCTAATGGTTAGCCTTTGCTTTC-3′ and reverse, 5′-CTAGCTAGCTATGGTCTCATGTCTGTGCAGG-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc., Otsu Japan), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min. .. The amplified fragment was inserted into the pYr-PromDetect vector (pLuc; Changsha Yingrun Biotechnology Co., Ltd., Changsha, China) and formed pYr-PromDetect-Cx32P vector (pLuc-Cx32P).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The complete coding sequence of GATA-3 was PCR amplified from human cDNA by using the following primers with additional NheI and XhoI digestion sites added (underlined respectively): Forward, 5′-CGCTAGCGATGGAGGTGACGGCGGA-3′ and reverse, 5′-GCCTCGAGCTAACCCATGGCGGTGAC-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc.), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min. .. The amplified fragment was inserted into a GV230 vector (pGv) and formed the pGATA3 vector.

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl. .. Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: Paragraph title: DNA extraction and polymerase chain reaction (PCR) amplification ... The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling
    Article Snippet: Paragraph title: Polymerase chain reaction (PCR) ... RT-PCR was performed in 0.2-mL tubes containing 10× Taq buffer, dNTP, Taq DNA polymerase (Takara, Japan), and 25 pmole of TPH1 primer (forward 5′-ATGATTGAAGACAATAAGGAG-3′ reverse 5′-AAGTTTTTGAGATACTCTCTG-3′) or GAPDH primer (forward 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse 5′-CAAAGTTGTCATGGATGACC-3′).

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: cDNAs encoding the complete open reading frame (ORF) of zebrafish mek1/2 and erk1 were obtained by polymerase chain reaction (PCR) amplification using gene-specific primers according to the NCBI’s zebrafish EST database. .. PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: End repair products were purified with the MinElute PCR purification kit (Qiagen, 28006). .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The PCR forward and reverse primers and product length were showed in following table . .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China). .. PCR undertook the following conditions: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30s, 57°C for 30s, and 72°C for 1min, and a final step of 72°C for 10min.

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: PCR forward and reverse primer sequences and product lengths are showed in Table . .. In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China). .. The PCR reaction was performed with the following conditions: initial denaturation at 94 C for 5 minutes; followed by 35 cycles at 94 C for 30 seconds, 55–58 C for 30 seconds for annealing, and 72 C for 45 seconds for elongation.

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: PCR forward and reverse primer sequences and product lengths are showed in Table . .. In brief, the PCR was performed in a 30 µL r eaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China). .. The PCR reaction was performed with the following conditions: initial denaturation at 94 C for 5 minutes; followed by 35 cycles at 94 C for 30 seconds, 55–58 C for 30 seconds for annealing, and 72 C for 45 seconds for elongation.

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: A total of 13 and 26 pairs of primers ( ) were designed to amplify all exons and their partial flanking intronic sequences based on the reference sequences of the bovine HSPA8 (NCBI Reference Sequence: AC_000172.1) and ERBB2 (NCBI Reference Sequence: AC_000176.1) referring to Bos_taurus_UMD_3.1 assembly using Primer3 web Program v.0.4.0 ( http://primer3.ut.ee ), respectively. .. The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). .. The PCR protocol was 5 min at 94 °C for initial denaturing, followed by 34 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min.

    Article Title: Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells
    Article Snippet: Reverse transcription was performed with a 20 μL total volume comprising 2 μL of extracted RNA, 1 μL of Takara Oligo dTl8 , 2 μL of Takara dNTP (10 mM), 0.5 μL of Takara RNasin, 1 μL of Takara AMV, 4 μL of 5× AMV Buffer, and 9.5 μL of DEPC-treated water. .. The qRT-PCR was performed in a 25 μL total volume comprising 2 μL of 10× PCR buffer, 2 μL of Takara dNTP (2.5 mM), 1 μL of 5′ primer (20 pmol/μL), 1 μL of 3′ primer (20 pmol/μL), 0.5 μL of Takara TaqE (5 U/μL), 2.5 μL of MgCl2 (25 mM), 11 μL of DEPC-treated water, and 5 μL of cDNA on the PTC-100 (MJ Research, Inc., Watertown, MA, USA). .. The sequences of the specific primers used in the research were as follows: caspase-3 – forward: 5′-AAACAGTATGCCGACAAG-3′ and reverse: 5′-GAGGGAAATACAGTACCAAA-3′; caspase-8 – forward: 5′-AAAGGGAACTTCAGACACC-3′ and reverse: 5′-CAGCAGGCTCTTGTTGAT-3′; caspase-9 – forward: 5′-CGAACTAACAGGCAAGCA-3′ and reverse: 5′-TTCACCTCCACCATGAAAT-3′; β-actin – forward: 5′-CGTCTGGACCTGGCTGGCCGGGACC-3′ and reverse: 5′-CATGAAGCATTTGCGGTGGACGATG-3′.

    Article Title: Enterovirus genotypes causing hand foot and mouth disease in Shanghai, China: a molecular epidemiological analysis
    Article Snippet: The reverse transcriptase reaction was carried out at 37°C for 30 min and 85°C for 5 s. Human enterovirus was preliminarily detected with highly conserved 5′UTR primers. .. The first PCR step was performed in a reaction volume of 25 μL, including 2 μL cDNA, 0.5 μmol each of outer primers (EV1F: 5′-CGGCCCCTGAATGCGGC-3′, EV1R: 5′-CACCGGATGGCCAATCCA-3′), 50 μmol dNTP, and 0.75U of ExTaq DNA polymerase (Takara, Dalian, China). .. The PCR protocol was to use an initial temperature of 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30s, 72°C for 30 s, and 72°C for 7 min. Then 1 μL of DNA from the first round of PCR was used as the template in the second round of PCR with the inner primers (EV2F: 5′-CCCCTGAATGCGGCTAAT-3′, EV2R: 5′-ATTGTCACCATAAGCAGCCA-3′) under the same reaction system and cycling conditions.

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: To simplify construction and utilize a strategy that did not rely on restriction enzymes, we used prolonged-overlap-PCR to replace the bgaB gene of pDL with gfpmut1 [ ]. .. Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. 2000 bp upstream of the start codon of the target protein were amplified as the promoter candidates.

    Article Title: Identification of a pathogenic mutation in a Chinese pedigree with polycystic kidney disease
    Article Snippet: As the PKD1 gene is complex, long-range PCR (LR-PCR) was performed to amplify PKD1 exons 22–26 (forward, 5′-TCCAGTCAAGTGGGCTCTC-3′, and reverse, 5′-CAATGAAGAGGAAAGCAGCAC-3′). .. In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total.

    Imaging:

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China). .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Sequencing:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. All first round PCRs were pooled and a fraction was used as template for the second round PCR.

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: All first round PCRs were pooled and a fraction was used as template for the second round PCR. .. The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 55C – 10 sec, 72C – 20 sec), 1x (68C – 2 min).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc., Otsu Japan), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min. .. The amplified fragment was inserted into the pYr-PromDetect vector (pLuc; Changsha Yingrun Biotechnology Co., Ltd., Changsha, China) and formed pYr-PromDetect-Cx32P vector (pLuc-Cx32P).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The complete coding sequence of GATA-3 was PCR amplified from human cDNA by using the following primers with additional NheI and XhoI digestion sites added (underlined respectively): Forward, 5′-CGCTAGCGATGGAGGTGACGGCGGA-3′ and reverse, 5′-GCCTCGAGCTAACCCATGGCGGTGAC-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc.), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min.

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Other sequencing libraries were constructed from 25 pg to 1 ng of ChIP DNA using TELP as described below. .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: A total of 13 and 26 pairs of primers ( ) were designed to amplify all exons and their partial flanking intronic sequences based on the reference sequences of the bovine HSPA8 (NCBI Reference Sequence: AC_000172.1) and ERBB2 (NCBI Reference Sequence: AC_000176.1) referring to Bos_taurus_UMD_3.1 assembly using Primer3 web Program v.0.4.0 ( http://primer3.ut.ee ), respectively. .. The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China).

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. After purification of the digested promoter candidates (E.Z.N.A.™ Cycle Pure Kit 200, Omega Bio-tek, Inc., USA) and the vector enzyme digestion products, the ligation was performed using T4 ligase (Thermo Fisher Scientific, USA) according to the instructions of the manufacturer and transformed into E . coli DH5α.

    Sonication:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: This step is only required for library construction from small amounts of DNA fragmented by mechanical shearing (e.g. sonication). .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Binding Assay:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer. .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    ChIP-sequencing:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Paragraph title: ChIP and high-sensitivity ChIP-seq library preparation ... Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    DNA Extraction:

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: Paragraph title: DNA extraction and polymerase chain reaction (PCR) amplification ... The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: Paragraph title: DNA Extraction and Genotyping ... The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: Paragraph title: DNA Extraction and Genotyping ... In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Identification of a pathogenic mutation in a Chinese pedigree with polycystic kidney disease
    Article Snippet: DNA extraction was performed using the DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocols. .. In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total.

    Nucleic Acid Electrophoresis:

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). .. The PCR protocol was 5 min at 94 °C for initial denaturing, followed by 34 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min.

    Fluorescence:

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl. .. The reverse transcripts obtained were used as PCR templates.

    RT Lamp Assay:

    Article Title: Rapid and Specific Detection of All Known Nipah virus Strains’ Sequences With Reverse Transcription-Loop-Mediated Isothermal Amplification
    Article Snippet: Paragraph title: RT-LAMP Assay ... The RT-LAMP reaction was performed in a 25.0 μl volume, containing 0.5 mM betaine (5 mM stock, Sigma-Aldrich Inc., St Louis, MO, United States), 1.4 mM each dNTP (10.0 mM stock, TaKaRa Bio Inc., Clontech Laboratories, Inc., Dalian, China), 2.5 μl of 10× ThermoPol Reaction Buffer [(1 × ThermoPol Reaction Buffer contains 20.0 mM Tris–HCl, pH 8.8; 10.0 mM KCl; 2.0 mM MgSO4 ; 10.0 mM (NH4 )2 SO4 ; 0.1% Triton X-100) New England Biolabs Inc.], 6.0 mM MgSO4 (New England Biolabs Inc.), 32 pM each of forward inner primer (FIP) and backward inner primer (BIP), 15 pM each of forward loop (LF) and backward loop (LB) primers (or replaced with an equal volume of nuclease-free water), 4 pM each of forward outer (F3) and backward outer (B3) primers, 8.0 U of Bst DNA polymerase large fragment (New England Biolabs Inc.), 7.5 U of WarmStart RTx reverse transcriptase (New England Biolabs Inc.; only added to the reaction system when the template was RNA), and 2.0 μl of pseudovirus RNA (≥50 pg/μl).

    Isolation:

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: Paragraph title: Isolation of the full-length mek1 , mek2 , and erk1 cDNAs of zebrafish ... PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan).

    Size-exclusion Chromatography:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech).

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 65C – 10 sec, 72C – 20 sec), 1x (68C – 2 min). .. The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The fragment of Cx32 promoter −651 to +84 (transcription start site as +1, total 735 base pairs) was PCR amplified from human genomic DNA by the following primers with restriction enzyme BglII and NheI sites added (underlined respectively): Forward, 5′-GAAGATCTAATGGTTAGCCTTTGCTTTC-3′ and reverse, 5′-CTAGCTAGCTATGGTCTCATGTCTGTGCAGG-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc., Otsu Japan), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min. .. The amplified fragment was inserted into the pYr-PromDetect vector (pLuc; Changsha Yingrun Biotechnology Co., Ltd., Changsha, China) and formed pYr-PromDetect-Cx32P vector (pLuc-Cx32P).

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: The complete coding sequence of GATA-3 was PCR amplified from human cDNA by using the following primers with additional NheI and XhoI digestion sites added (underlined respectively): Forward, 5′-CGCTAGCGATGGAGGTGACGGCGGA-3′ and reverse, 5′-GCCTCGAGCTAACCCATGGCGGTGAC-3′. .. The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc.), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min. .. The amplified fragment was inserted into a GV230 vector (pGv) and formed the pGATA3 vector.

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl. .. The total PCR mixture consisted of 10 µl SYBR 2X Green I fluorescence reaction fluid (Beijing Bioteke Biological Technology Co., Ltd., Beijing, China), 0.25 µl of each of the forward and reverse primers (10 µmol/l; forward, 5′-CCATACCACCCTGAACGC-3′ and reverse, 5′-AGCCTACAGCACCCGGTAT-3′), 1 µl sample template and RNase free water, to give a total reaction volume of 20 µl.

    Article Title: Identification of a pathogenic mutation in a Chinese pedigree with polycystic kidney disease
    Article Snippet: In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total. .. In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total.

    Purification:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: End repair products were purified with the MinElute PCR purification kit (Qiagen, 28006). .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer. .. The enzyme digestion sites in the vector 5’ and 3’ ends were BamHI and KpnI (BamHI/EcoRI, EcoRI/KpnI).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling
    Article Snippet: Primers for GAPDH were supplied by Qiagen, CA, USA. .. RT-PCR was performed in 0.2-mL tubes containing 10× Taq buffer, dNTP, Taq DNA polymerase (Takara, Japan), and 25 pmole of TPH1 primer (forward 5′-ATGATTGAAGACAATAAGGAG-3′ reverse 5′-AAGTTTTTGAGATACTCTCTG-3′) or GAPDH primer (forward 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse 5′-CAAAGTTGTCATGGATGACC-3′). .. Amplified PCR products were separated in 1.5% agarose gels, visualized using a gel imaging system (UVP, Cambridge, UK), and quantitated by measuring band densities.

    Construct:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Other sequencing libraries were constructed from 25 pg to 1 ng of ChIP DNA using TELP as described below. .. Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Article Title: Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle
    Article Snippet: A DNA pool was constructed from aforementioned 17 Holstein bulls (50 ng/uL of each individual) to identify potential SNPs that were involved in HSPA8 and ERBB2 genes. .. The polymerase chain reaction (PCR) was performed to amplify the pooled DNA from 17 sires with a final reaction volume of 25 μL, comprising of 50 ng genomic DNA, 0.5 μL of each primer, 2.5 μL 10× PCR buffer, 2.5 mM each of dNTP, and 1 U of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China).

    Chromatin Immunoprecipitation:

    Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing
    Article Snippet: Paragraph title: ChIP and high-sensitivity ChIP-seq library preparation ... Subsequently, 1 μl of terminal deoxynucleotidyl transferase (TDT; NEB, M0315S) was added, and the reaction mixture was incubated at 37°C for 35 min. After poly-C tailing, TDT was inactivated by heating to 75°C for 20 min. An extension step was performed by adding the following extension mix to the above-mentioned TDT reaction: 6.2 μl of H2 O; 0.8 μl of KAPA2G Robust HS (KAPA, KK5515); 12 μl of 5× KAPA buffer A (KAPA, supplied with enzyme); 4.8 μl of 2.5 mM dNTP (Takara, supplied with RR006A) and 6 μl of 2 μM biotin-labeled anchor primer.

    Plasmid Preparation:

    Article Title: Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis
    Article Snippet: Paragraph title: Construction of pYr-PromDetect-Cx32P vector ... The PCR reaction was performed using PrimeSTAR-HS DNA polymerase in PCR buffer containing 2.5 mM dNTP (Takara Bio, Inc., Otsu Japan), with the thermocycling condition: 94°C for 2 min, then 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, for 30 cycles, followed by 72°C for 3 min.

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Article Title: Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications
    Article Snippet: Paragraph title: Construction of the promoter-probe vector and vectors carrying different promoter candidates and pMA5 containing PtrnQ ... Promoter candidate fragments were prepared by PCR amplification using B . subtilis 168 genomic DNA as template, PrimerSTAR Max DNA Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and primers (Genwiz, China) listed in , according to the instructions of the manufacturer.

    Real-time Polymerase Chain Reaction:

    Article Title: MiR-494 acts as a tumor promoter by targeting CASP2 in non-small cell lung cancer
    Article Snippet: Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl. .. Primer, DEPC water, and RNA were first incubated at 70 °C for 10 minutes, followed by dNTP, buffer, reverse transcriptase at 30 °C for 10 minutes, 42 °C for 60 minutes, and 70 °C for 10 minutes.

    Article Title: Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells
    Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction (qRT-PCR) ... The qRT-PCR was performed in a 25 μL total volume comprising 2 μL of 10× PCR buffer, 2 μL of Takara dNTP (2.5 mM), 1 μL of 5′ primer (20 pmol/μL), 1 μL of 3′ primer (20 pmol/μL), 0.5 μL of Takara TaqE (5 U/μL), 2.5 μL of MgCl2 (25 mM), 11 μL of DEPC-treated water, and 5 μL of cDNA on the PTC-100 (MJ Research, Inc., Watertown, MA, USA).

    RNA Extraction:

    Article Title: MicroRNA differential expression spectrum and microRNA-125a-5p inhibition of laryngeal cancer cell proliferation
    Article Snippet: A total of 2 ng to 2 µg RNA from each total RNA extraction was used for RT-qPCR. .. Following this, 2 µl RNase inhibitor, 8 µl M-MLV buffer (5X), 2 µl M-MLV reverse transcriptase (RNase H− ) and 2 µl dNTP (Takara Bio, Otsu, Japan) were added to the reaction mixture and RNase-free water was added to reach a total reaction volume of 40 µl.

    Article Title: MiR-494 acts as a tumor promoter by targeting CASP2 in non-small cell lung cancer
    Article Snippet: Paragraph title: RNA extraction and quantitative RT-PCR ... Reactions contained 4 µl of 5 X buffer, 1 µl of 10 mmol/L (mM) dNTP, and 0.5 µl of reverse transcriptase (TaKaRa, Japan); DEPC water was added up to a total volume of 20 µl.

    Agarose Gel Electrophoresis:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). .. PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 55C – 10 sec, 72C – 20 sec), 1x (68C – 2 min).

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template. .. The PCR was conducted on a Mastercycler Gradient PCR thermal cycler (Eppendorf, Hamburg, Germany), with the following conditions: Denaturation for 15 min at 95°C, 50 cycles of annealing at 95°C for 30 sec, 57°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 5 min.

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China). .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China). .. In brief, the PCR was performed in a 30 µL reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Genetic variants of the dUTPase-encoding gene DUT increase HR-HPV infection rate and cervical squamous cell carcinoma risk
    Article Snippet: In brief, the PCR was performed in a 30 µL r eaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China). .. In brief, the PCR was performed in a 30 µL r eaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.5 units of Taq DNA polymerase (TAKARA, Dalian, China).

    Article Title: Enterovirus genotypes causing hand foot and mouth disease in Shanghai, China: a molecular epidemiological analysis
    Article Snippet: The first PCR step was performed in a reaction volume of 25 μL, including 2 μL cDNA, 0.5 μmol each of outer primers (EV1F: 5′-CGGCCCCTGAATGCGGC-3′, EV1R: 5′-CACCGGATGGCCAATCCA-3′), 50 μmol dNTP, and 0.75U of ExTaq DNA polymerase (Takara, Dalian, China). .. The PCR protocol was to use an initial temperature of 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30s, 72°C for 30 s, and 72°C for 7 min. Then 1 μL of DNA from the first round of PCR was used as the template in the second round of PCR with the inner primers (EV2F: 5′-CCCCTGAATGCGGCTAAT-3′, EV2R: 5′-ATTGTCACCATAAGCAGCCA-3′) under the same reaction system and cycling conditions.

    Electrophoresis:

    Article Title: Enterovirus genotypes causing hand foot and mouth disease in Shanghai, China: a molecular epidemiological analysis
    Article Snippet: The first PCR step was performed in a reaction volume of 25 μL, including 2 μL cDNA, 0.5 μmol each of outer primers (EV1F: 5′-CGGCCCCTGAATGCGGC-3′, EV1R: 5′-CACCGGATGGCCAATCCA-3′), 50 μmol dNTP, and 0.75U of ExTaq DNA polymerase (Takara, Dalian, China). .. The PCR protocol was to use an initial temperature of 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30s, 72°C for 30 s, and 72°C for 7 min. Then 1 μL of DNA from the first round of PCR was used as the template in the second round of PCR with the inner primers (EV2F: 5′-CCCCTGAATGCGGCTAAT-3′, EV2R: 5′-ATTGTCACCATAAGCAGCCA-3′) under the same reaction system and cycling conditions.

    Preserving:

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: All DNA samples were dissolved in water and hypothermic preservation ready to use. .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

    Spectrophotometry:

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: The purity of the DNA was assessed by the A260/A280 ratio, and the DNA concentration was calculated from the absorption readings obtained from a spectrophotometer (DU 800; Beckman Coulter, Inc., Brea, CA, USA). .. The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Concentration Assay:

    Article Title: H19 gene methylation status is associated with male infertility
    Article Snippet: The purity of the DNA was assessed by the A260/A280 ratio, and the DNA concentration was calculated from the absorption readings obtained from a spectrophotometer (DU 800; Beckman Coulter, Inc., Brea, CA, USA). .. The total volume of the fundamental reaction system was 25 µl, which consisted of 0.25 µl LA Taq polymerase, 2.5 µl 10X LA Taq buffer, 4 µl dNTP (all Takara Biotechnology Co., Ltd., Dalian, China), 3.25 µl deionized water, 5 µl sense primer (2 µM), 5 µl antisense primer (2 µM), and 5 µl DNA template.

    Article Title: Identification of a pathogenic mutation in a Chinese pedigree with polycystic kidney disease
    Article Snippet: Following determination of the size distribution and concentration of the fragments, the DNA library was sequenced on the Illumina HiSeq 4000 platform for paired-end 150 bp reads. .. In the LR-PCR system, 100 ng template, 2 µl dNTP (4019; Takara Bio, Inc., Otsu, Japan), 10 µl 2XGC buffer (9155; Takara Bio, Inc.), 1 µl forward primer (10 pmol/l), 1 µl reverse primer (10 pmol/l), 0.2 µl LA Polymerase (RR02MA; Takara Bio, Inc.) and water were used: 20 µl in total.

    CTG Assay:

    Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
    Article Snippet: cDNAs encoding the complete open reading frame (ORF) of zebrafish mek1/2 and erk1 were obtained by polymerase chain reaction (PCR) amplification using gene-specific primers according to the NCBI’s zebrafish EST database. .. PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2 , 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). .. Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany).

    Staining:

    Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population
    Article Snippet: The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China). .. The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China).

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    TaKaRa dntps
    Transcription- and translation-coupled DNA <t>(TTcDR)</t> replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including <t>dNTPs,</t> yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
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    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    Extension reactions with mixed dNTPs. ( A ) Sequences of 5′-FAM labeled 18-nt primer and 26-nt templates. ( B ) PAGE analysis of extension reactions using the natural and oxidized dNTPs (each dNTP at 10 µM) in the natural template. Lane 1 refers to the appropriate size markers. ( C ) Extension reactions in the presence of dGTP, dTTP, dATP and dC o TP (each dNTP at 10 µM) were shown in lanes 2 and 3 by KF (exo − ) and lanes 6 and 7 by Vent (exo − ). Extension reactions in the presence of dGTP, dTTP, dCTP and dA o TP (each dNTP at 10 µM) were shown in lanes 4 and 5 by KF (exo − ) and lanes 8 anmd 9 by Vent (exo − ). Lane 1 refer to the appropriate marker.

    Journal: Nucleic Acids Research

    Article Title: Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

    doi: 10.1093/nar/gkq914

    Figure Lengend Snippet: Extension reactions with mixed dNTPs. ( A ) Sequences of 5′-FAM labeled 18-nt primer and 26-nt templates. ( B ) PAGE analysis of extension reactions using the natural and oxidized dNTPs (each dNTP at 10 µM) in the natural template. Lane 1 refers to the appropriate size markers. ( C ) Extension reactions in the presence of dGTP, dTTP, dATP and dC o TP (each dNTP at 10 µM) were shown in lanes 2 and 3 by KF (exo − ) and lanes 6 and 7 by Vent (exo − ). Extension reactions in the presence of dGTP, dTTP, dCTP and dA o TP (each dNTP at 10 µM) were shown in lanes 4 and 5 by KF (exo − ) and lanes 8 anmd 9 by Vent (exo − ). Lane 1 refer to the appropriate marker.

    Article Snippet: DNA polymerase I Klenow fragment (exo+ ), Pyrobest polymerase, and dNTPs were purchased from Takara Bio, Inc. Vent (exo− ) DNA polymerase was purchased from New England Biolabs, Inc. ODNs used in the enzyme reactions and T m experiments were purchased from Sigma-Aldrich Japan.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Marker

    Extension reactions with mixed dNTPs. ( A ) Sequences of 5′-FAM labeled 18-nt primer and 26-nt templates. ( B ) PAGE analysis of extension reactions using mixed dNTPs (each dNTP at 10 µM) in the oxidized templates. Lanes 1 refers to the appropriate marker. ( C ) PAGE analysis of extension reactions in the presence of dGTP, dTTP, dATP and dCTP (each dNTP at 10 µM). Lane 1 refer to the appropriate marker. Extension reactions by KF (exo − ) are shown in lanes 2–5, and its reactions by Vent (exo − ) are shown in lanes 6–9.

    Journal: Nucleic Acids Research

    Article Title: Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

    doi: 10.1093/nar/gkq914

    Figure Lengend Snippet: Extension reactions with mixed dNTPs. ( A ) Sequences of 5′-FAM labeled 18-nt primer and 26-nt templates. ( B ) PAGE analysis of extension reactions using mixed dNTPs (each dNTP at 10 µM) in the oxidized templates. Lanes 1 refers to the appropriate marker. ( C ) PAGE analysis of extension reactions in the presence of dGTP, dTTP, dATP and dCTP (each dNTP at 10 µM). Lane 1 refer to the appropriate marker. Extension reactions by KF (exo − ) are shown in lanes 2–5, and its reactions by Vent (exo − ) are shown in lanes 6–9.

    Article Snippet: DNA polymerase I Klenow fragment (exo+ ), Pyrobest polymerase, and dNTPs were purchased from Takara Bio, Inc. Vent (exo− ) DNA polymerase was purchased from New England Biolabs, Inc. ODNs used in the enzyme reactions and T m experiments were purchased from Sigma-Aldrich Japan.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Marker